Method and probe set for detecting cancer

ABSTRACT

Methods for detecting cancer that include hybridizing a set of chromosomal probes to a biological sample obtained from a patient, and identifying if aneusomic cells are present in a selected subset of cells obtained from the biological sample are described. A set of chromosomal probes and kits for detecting cancer that include sets of chromosomal probes, are also described.

TECHNICAL FIELD

[0001] The invention relates to detecting cancer.

BACKGROUND OF THE INVENTION

[0002] Bladder cancer represents the fifth most common neoplasm and thetwelfth leading cause of cancer death in the United States, where over53,000 new cases are diagnosed each year. Over 95% of bladder cancercases in the United States are transitional cell carcinoma (TCC,sometimes referred to as urothelial cell carcinoma). Tumor stage is thebest predictor of prognosis for patients with bladder cancer. Bladdercancer is staged according to the depth of invasion of the tumor andwhether or not there are lymph node or distant metastases. Non-invasivepapillary tumors (the most common and least aggressive type of bladdertumor) are referred to as stage pTa tumors. “Flat” TCC, more commonlyreferred to as “carcinoma in situ” (CIS) is a more aggressive but lesscommon tumor that is associated with a high rate of progression toinvasive disease. CIS is assigned a stage of pTIS. Tumors that haveinvaded through the basement membrane of the epithelium into theunderlying lamina propria are assigned a stage of pT1. A tumor that hasinvaded the muscle of the bladder is a stage pT2 tumor. Invasion throughthe muscle into the tissue surrounding the bladder is a pT3 tumor.Invasion into surrounding organs is a pT4 tumor. The term “superficial”bladder cancer refers to pTa, pTIS, and pT1 tumors. Muscle-invasivebladder cancer refers to pT2, pT3, and pT4 tumors.

[0003] Approximately 80% of bladder cancer cases present as“superficial” bladder cancer and the remaining 20% as muscle-invasivebladder cancer. Patients with “superficial” bladder cancer do notrequire cystectomy (i.e. removal of the bladder) but have a high risk oftumor recurrence, and are monitored for tumor recurrence and/progressionon a regular basis (usually every 3 months for the first 2 years, every6 months for the next 2 years, and every year thereafter). Treatment forsuperficial bladder cancer generally consists of surgical removal ofpapillary tumors and treatment of CIS with Bacillus-Calmette Guerin(BCG). Patients with muscle invasive disease are treated by cystectomyand have a relatively poor prognosis compared to patients with“superficial” bladder cancer. Unfortunately, 80-90% of patients withmuscle invasive bladder cancer initially present with muscle invasivedisease. A large share of the estimated 10,000 deaths per year frombladder cancer is accounted for by this group of patients. The fact thatmany patients with advanced bladder cancer present that way suggeststhat screening programs that detect bladder cancer at earlier stages mayhelp reduce the overall mortality from the disease. In fact, at leasttwo large screening studies suggest that screening does help identifybladder cancer at earlier stages. Messing et al., Urology, 45:387-396,1995; and Mayfield and Whelan, Br. J. Urol., 82(6):825-828, 1998.

[0004] Cystoscopy and urine cytology have been the mainstays for bladdercancer detection over the past several decades. Several studies,however, have shown that cytology has a disappointingly low sensitivityfor bladder cancer detection. Mao et al., Science, 271:659-662, 1996;Ellis et al., Urology, 50:882-887, 1997; and Landman et al., Urology,52:398-402, 1998. For this reason, there has been great interest in thedevelopment of new assays that have increased sensitivity for thedetection of bladder cancer. Examples of new assays that have beendeveloped for bladder cancer detection include tests that detect bladdertumor antigens, e.g. BT test (C. R. Bard, Inc., Murrayhill, N.J.),NMP-22, FDP, etc., tests that detect increased telomerase activity(usually associated with malignancy), or tests that detect geneticalterations in urinary cells and bladder washings (e.g. fluorescence insitu hybridization (FISH) and microsatellite analysis). Although FISHanalysis may be more sensitive than other detection methods, largenumbers of cells must be counted, and consequently, the analysis is timeconsuming and costly. Therefore, a need exists for a rapid method ofdetecting cancer that maintains adequate sensitivity.

SUMMARY OF THE INVENTION

[0005] The invention is based, in part, on the discovery that a rapid,sensitive method for detecting cancer can be based on the presence ofaneusomic cells in a selected subset of cells from a biological sample.Selection of a subset of cells to be evaluated for chromosomal anomaliesreduces the number of cells to be analyzed, allowing analysis to beperformed in a rapid manner while maintaining, and even improving,sensitivity. The invention also provides a set of chromosomal probesselected to provide the optimal sensitivity in FISH analysis and kitsfor detecting cancer that include sets of chromosomal probes.

[0006] In one aspect, the invention features a method of screening forcancer in a subject. The method includes the steps of hybridizing a setof chromosomal probes to a biological sample from the subject; selectingcells from the biological sample; determining the presence or absence ofaneusomic cells in the selected cells; and correlating the presence ofaneusomic cells in the selected cells with cancer in the subject. Thebiological sample can be urine, blood, cerebrospinal fluid, pleuralfluid, sputum, peritoneal fluid, bladder washings, oral washings, tissuesamples, touch preps, or fine-needle aspirates, and can be concentratedprior to use. Urine is a particularly useful biological sample. Thecells can be selected by nuclear morphology including nucleus size andshape. Nuclear morphology can be assessed by DAPI staining. The methodis useful for detecting cancers such as bladder cancer, lung cancer,breast cancer, ovarian cancer, prostate cancer, colorectal cancer, renalcancer, and leukemia. The method is particularly suited for detectingbladder cancer.

[0007] The set of chromosomal probes includes at least three chromosomalprobes. The set can include at least one centromeric probe or at leastone locus specific probe. Suitable centromeric chromosomal probesinclude probes to chromosomes 3, 7, 8, 11, 15, 17, 18, and Y. A suitablelocus specific probe includes a probe to the 9p21 region of chromosome9. For example, the set can include centromeric chromosomal probes 3, 7,and 17, and further can include locus specific probe 9p21. Thechromosomal probes can be fluorescently labeled.

[0008] The invention also features sets of chromosomal probes and kitsfor detecting cancer that include sets of chromosomal probes, thatinclude centromeric probes to chromosomes 3, 7, and 17, and further caninclude a locus-specific probe such as 9p21. The chromosomal probes canbe fluorescently labeled.

[0009] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used topractice the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

[0010] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION

[0011] The invention advantageously provides a rapid, sensitive methodfor detecting cancer, and can be used to screen subjects at risk forcancer, including solid tumors and leukemias, or to monitor patientsdiagnosed with cancer for tumor recurrence. For example, subjects atrisk for bladder cancer, lung cancer, breast cancer, ovarian cancer,prostate cancer, colorectal cancer, head and neck cancer, renal cancer,or leukemia can be screened or monitored for recurrence. In general, aset of chromosomal probes is hybridized to cells (from urine or otherbiological sample) on a slide. The cells on the slide are then visuallyscanned at a relatively low power (e.g. 200-400×) for morphologicfeatures strongly suggestive of malignancy (e.g. increased nuclear sizeor irregular nuclear shape). The nuclei of the cytologically abnormalcells are then examined for chromosomal abnormalities by switching theobjective to a higher power (e.g. 600-1000×) and “flipping” the filtersto determine if the cell is aneusomic or not. Use of this processmarkedly reduces the time spent assessing cells that have a lowprobability of being neoplastic and allows the examiner to focus theirefforts on the cells that have a much higher probability of beingneoplastic and showing aneusomy.

[0012] In Situ Hybridization

[0013] The presence or absence of aneusomic cells is determined by insitu hybridization. “Aneusomic cells” are cells having an abnormalnumber of chromosomes or having chromosomal structural alterations suchas hemizygous or homozygous loss of a specific chromosomal region.Typically, aneusomic cells having one or more chromosomal gains, i.e.,three or more copies of any given chromosome, are considered testpositive in the methods described herein, although cells exhibitingmonosomy and nullisomy also may be considered test positive undercertain circumstances. In general, in situ hybridization includes thesteps of fixing a biological sample, hybridizing a chromosomal probe totarget DNA contained within the fixed biological sample, washing toremove non-specific binding, and detecting the hybridized probe.

[0014] A “biological sample” is a sample that contains cells or cellularmaterial. Typically, the biological sample is concentrated prior tohybridization to increase cell density. Non-limiting examples ofbiological samples include urine, blood, cerebrospinal fluid (CSF),pleural fluid, sputum, and peritoneal fluid, bladder washings,secretions (e.g. breast secretion), oral washings, tissue samples, touchpreps, or fine-needle aspirates. The type of biological sample that isused in the methods described herein depends on the type of cancer onewishes to detect. For example, urine and bladder washings provide usefulbiological samples for the detection of bladder cancer and to a lesserextent prostate or kidney cancer. Pleural fluid is useful for detectinglung cancer, mesothelioma or metastatic tumors (e.g. breast cancer), andblood is a useful biological sample for detecting leukemia. For tissuesamples, the tissue can be fixed and placed in paraffin for sectioning,or frozen and cut into thin sections.

[0015] Typically, cells are harvested from a biological sample usingstandard techniques. For example, cells can be harvested by centrifuginga biological sample such as urine, and resuspending the pelleted cells.Typically, the cells are resuspended in phosphate-buffered saline (PBS).After centrifuging the cell suspension to obtain a cell pellet, thecells can be fixed, for example, in acid alcohol solutions, acid acetonesolutions, or aldehydes such as formaldehyde, paraformaldehyde, andglutaraldehyde. For example, a fixative containing methanol and glacialacetic acid in a 3:1 ratio, respectively, can be used as a fixative. Aneutral buffered formalin solution also can be used, and includesapproximately 1% to 10% of 37-40% formaldehyde in an aqueous solution ofsodium phosphate. Slides containing the cells can be prepared byremoving a majority of the fixative, leaving the concentrated cellssuspended in only a portion of the solution.

[0016] The cell suspension is applied to slides such that the cells donot overlap on the slide. Cell density can be measured by a light orphase contrast microscope. For example, cells harvested from a 20 to 100ml urine sample typically are resuspended in a final volume of about 100to 200 μl of fixative. Three volumes of this suspension (usually 3, 10,and 30 μl), are then dropped into 6mm wells of a slide. The cellularity(i.e. density of cells) in these wells is then assessed with a phasecontrast microscope. If the well containing the greatest volume of cellsuspension does not have enough cells, the cell suspension isconcentrated and placed in another well.

[0017] Prior to in situ hybridization, chromosomal probes andchromosomal DNA contained within the cell each are denatured.Denaturation typically is performed by incubating in the presence ofhigh pH, heat (e.g., temperatures from about 70° C. to about 95° C.),organic solvents such as formamide and tetraalkylammonium halides, orcombinations thereof. For example, chromosomal DNA can be denatured by acombination of temperatures above 70° C. (e.g., about 73° C.) and adenaturation buffer containing 70% formamide and 2×SSC (0.3M sodiumchloride and 0.03 M sodium citrate). Denaturation conditions typicallyare established such that cell morphology is preserved. Chromosomalprobes can be denatured by heat. For example, probes can be heated toabout 73° C. for about five minutes.

[0018] After removal of denaturing chemicals or conditions, probes areannealed to the chromosomal DNA under hybridizing conditions.“Hybridizing conditions” are conditions that facilitate annealingbetween a probe and target chromosomal DNA. Hybridization conditionsvary, depending on the concentrations, base compositions, complexities,and lengths of the probes, as well as salt concentrations, temperatures,and length of incubation. The higher the concentration of probe, thehigher the probability of forming a hybrid. For example, in situhybridizations are typically performed in hybridization buffercontaining 1-2×SSC, 50% formamide and blocking DNA to suppressnon-specific hybridization. In general, hybridization conditions, asdescribed above, include temperatures of about 25° C. to about 55° C.,and incubation lengths of about 0.5 hours to about 96 hours. Moreparticularly, hybridization can be performed at about 32° C. to about40° C. for about 2 to about 16 hours.

[0019] Non-specific binding of chromosomal probes to DNA outside of thetarget region can be removed by a series of washes. Temperature andconcentration of salt in each wash depend on the desired stringency. Forexample, for high stringency conditions, washes can be carried out atabout 65° C. to about 80° C., using 0.2× to about 2×SSC, and about 0.1%to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40).Stringency can be lowered by decreasing the temperature of the washes orby increasing the concentration of salt in the washes.

[0020] Chromosomal Probes

[0021] Suitable probes for in situ hybridization in accordance with theinvention hybridize (i.e., form a duplex) with repetitive DNA associatedwith the centromere of a chromosome. Centromeres of primate chromosomescontain a complex family of long tandem repeats of DNA, composed of amonomer repeat length of about 171 base pairs, that is referred to asalpha-satellite DNA. Non-limiting examples of centromeric chromosomalprobes include probes to chromosomes 3, 7, 8, 11, 15, 17, 18, and Y.Locus-specific probes that hybridize to a critical chromosomal region,such as the 9p21 region of chromosome 9, also are suitable.

[0022] Chromosomal probes are chosen for maximal sensitivity andspecificity. Using a set of chromosomal probes (i.e., two or moreprobes) provides greater sensitivity and specificity than use of any onechromosomal probe. Thus, based on the results herein, chromosomal probesthat detect the most frequently aneusomic chromosomes, and thatcomplement each other, are included in a set. For example, based ondiscrimination values of probes determined herein, a set of chromosomalprobes can include centromeric probes to chromosomes 3, 7, and 17.Additionally, the set can include probes to the 9p21 region ofchromosome 9 or a centromeric probe to chromosome 8, chromosome 9,chromosome 11, or chromosome 18. As described herein, a probe tochromosome 7 when used alone demonstrated a high sensitivity, and coulddetect about 76% of bladder cancers. Probes to chromosomes 3 and 17, andto the 9p21 region of chromosome 9 were able to detect additionalbladder cancer cases that showed no abnormality with chromosome 7 probealone. The combination of probes to chromosomes 3, 7, 17, and to 9p21provide a sensitivity of about 95% for detecting bladder cancer in thecohort of patients described herein.

[0023] Chromosomal probes are typically about 50 to about 1×10⁵nucleotides in length. Longer probes typically comprise smallerfragments of about 100 to about 500 nucleotides in length. Probes thathybridize with centromeric DNA and locus-specific DNA are availablecommercially, for example, from Vysis, Inc. (Downers Grove, Ill.),Molecular Probes, Inc. (Eugene, Oreg.), or from Cytocell (Oxfordshire,UK). Alternatively, probes can be made non-commercially from chromosomalor genomic DNA through standard techniques. For example, sources of DNAthat can be used include genomic DNA, cloned DNA sequences, somatic cellhybrids that contain one, or a part of one, human chromosome along withthe normal chromosome complement of the host, and chromosomes purifiedby flow cytometry or microdissection. The region of interest can beisolated through cloning, or by site-specific amplification via thepolymerase chain reaction (PCR). See, for example, Nath and Johnson,Biotechnic Histochem., 1998, 73(1):6-22, Wheeless et al., Cytometry.1994, 17:319-326, and U.S. Pat. No. 5,491,224.

[0024] Chromosomal probes typically are directly labeled with afluorophore, an organic molecule that fluorescesces after absorbinglight of lower wavelength/higher energy. The fluorophore allows theprobe to be visualized without a secondary detection molecule. Aftercovalently attaching a fluorophore to a nucleotide, the nucleotide canbe directly incorporated into the probe with standard techniques such asnick translation, random priming, and PCR labeling. Alternatively,deoxycytidine nucleotides within the probe can be transaminated with alinker. The fluorophore then is covalently attached to the transaminateddeoxycytidine nucleotides. See, U.S. Pat. No. 5,491,224.

[0025] Fluorophores of different colors are chosen such that eachchromosomal probe in the set can be distinctly visualized. For example,a combination of the following fluorophores may be used:7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Redsm (MolecularProbes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, lissaminerhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate(FITC), 7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and Cascade™blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.). Probes areviewed with a fluorescence microscope and an appropriate filter for eachfluorophore, or by using dual or triple band-pass filter sets to observemultiple fluorophores. See, for example, U.S. Pat. No. 5,776,688.Alternatively, techniques such as flow cytometry can be used to examinethe hybridization pattern of the chromosomal probes.

[0026] Probes also can be indirectly labeled with biotin or digoxygenin,or labeled with radioactive isotopes such as ³²P and ³H, althoughsecondary detection molecules or further processing then is required tovisualize the probes. For example, a probe indirectly labeled withbiotin can be detected by avidin conjugated to a detectable marker. Forexample, avidin can be conjugated to an enzymatic marker such asalkaline phosphatase or horseradish peroxidase. Enzymatic markers can bedetected in standard calorimetric reactions using a substrate and/or acatalyst for the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

[0027] Selection of Cells

[0028] According to the invention, cells are microscopically selectedfrom the cells of a biological sample (e.g. urine, etc.) on a slideprior to assessing if aneusomic cells are present or absent. “Selecting”refers to the identification of cells that are more likely to beneoplastic due to one or more cytologic (mainly nuclear) abnormalitiessuch as nuclear enlargement, nuclear irregularity or abnormal nuclearstaining (usually a mottled staining pattern). These nuclear features,can be assessed with nucleic acid stains or dyes such as propidiumiodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Propidiumiodide is a red-fluorescing DNA-specific dye that can be observed at anemission peak wavelength of 614 nm. Typically, propidium iodide is usedat a concentration of about 0.4 μg/ml to about 5 μg/ml. DAPI, a bluefluorescing DNA-specific stain that can be observed at an emission peakwavelength of 452 nm, generally is used at a concentration ranging fromabout 125 ng/ml to about 1000 ng/ml. Staining of cells with DAPI orpropidium iodide is generally performed after in situ hybridization isperformed.

[0029] Determining Presence of Aneusomic Cells

[0030] After a cell is selected based on one or more of the statedcriteria, the presence or absence of aneusomy is assessed by examiningthe hybridization pattern of the chromosomal probes (i.e. the number ofsignals for each probe) in each selected cell, and recording the numberof chromosome signals. This step is repeated until the hybridizationpattern has been assessed in at least 4 cells, if all 4 cells areaneusomic. In a typical assay, the hybridization pattern is assessed inabout 20 to about 25 selected cells.

[0031] Cells with more than two copies of multiple chromosomes (i.e.,gains of multiple chromosomes) are considered cancer positive. Samplescontaining about 20 selected cells and at least about 4 test positivecells typically are considered cancer positive. If less than about 4test positive cells are found, the level of chromosome ploidy isdetermined. A cancer positive result also is indicated if more than 30%of the cells demonstrate hemizygous or homozygous loss (i.e., nullisomy)of a specific chromosome region, such as loss of 9p21 in bladder cancer.Nullisomy can be confirmed-as non-artifactual by observing thesurrounding normal appearing cells to see if they have two signals forthe specific chromosomal region.

[0032] Screening and Monitoring Patients for Cancer

[0033] The methods described herein can be used to screen patients forcancer, or can be used to monitor patients diagnosed with cancer. Forexample, in a screening mode, patients at risk for bladder cancer, suchas patients older than 50 who smoke, or patients chronically exposed toaromatic amines, are screened with the goal of earlier detection ofbladder cancer. The methods described herein can be used alone, or inconjunction with other tests, such as the hemoglobin dipstick test. Forexample, a patient having an increased risk of bladder cancer can bescreened for bladder cancer by detecting hemoglobin in the urine, i.e.,hematuria. During such a screening process, patients without hematuriado not need further analysis, and are instead, re-examined for hematuriain an appropriate amount of time, e.g., at their annual check-up.Samples from patients with hematuria are further analyzed using themethods described herein. In general, a set of chromosomal probes ishybridized with the biological sample, a subset of cells is selected,and the presence of aneusomic cells is determined in the selected cells.Patients that have aneusomic cells are further examined, for example, bycystoscopy, and can receive appropriate treatment, if necessary. Aftertreatment, patients are monitored for cancer recurrence using themethods described herein.

[0034] The superior sensitivity of the methods described herein indicatethat this technique could replace cytology for the detection andmonitoring of cancers such as bladder cancer. The majority of patientswith bladder cancer will have detectable aneusomic cells and can bemonitored for treatment efficacy, and tumor recurrence/progression withthe methods described herein. A small proportion of patients withcystoscopic or biopsy evidence of bladder cancer (primarily those withlow grade-non-invasive tumors) may not have detectable aneusomic cellsin their urine. These patients (i.e. those with low grade papillarytumors) are at very low rate of tumor progression and may beconveniently monitored by a combination of the methods described hereinand cystoscopy. The appearance of anuesomic cells in the urine of thesepatients may herald the development of a more aggressive tumor in thissubset of patients. The high sensitivity and specificity of the FISHtest described herein for aggressive bladder cancers may help reduce thefrequency of cystoscopy.

[0035] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Samples and Sample Preparation

[0036] Samples included voided urine from 21 biopsy proven bladdercancer cases, in which a diagnosis was made by either positive cytologyor by histology, in the case of cytology negative samples. Control urinesamples included nine samples from normal healthy donors (age 25-80),and three samples from patients with genitourinary diseases other thanbladder cancer.

[0037] Approximately 50 to 200 ml of urine were collected per patient.Urine samples were stored at 4° C. for less than 48 hours, and processedby centrifugation at 1200 g for 5 minutes. The supematant was discarded,and the pellet resuspended in 10 ml of 0.075 M KCl, and incubated atroom temperature for 15 minutes. Samples were spun for 5 minutes at 1200g, and the KCl solution was removed. Pellets were resuspended in 10 mlof a 3:1 methanol:glacial acetic acid fixative, and centrifuged for 8minutes at 1200 g. The fixative was carefully removed leaving the cellpellet, and this step was repeated two more times.

[0038] Density of the slides was monitored by frequently checking itunder a phase contrast microscope, using a 20× objective, betweendroppings. Generally, it was attempted to obtain as many cells aspossible on the slide without having any cell overlap. If a samplecontained low numbers of cells, as much of the sample as possible wasplaced on the slide. In samples with very low numbers of cells, thewhole sample was used. Slides were dried overnight at room temperature.

[0039] Slides containing the samples were incubated in 2×SSC at 37° C.for 10-30 minutes, then incubated in 0.2 mg/ml pepsin for 20 minutes at37° C. Slides then were washed in PBS twice, for 2 minutes per wash, atroom temperature. Cells were fixed in 2.5% Neutral Buffered Formalin for5 minutes at room temperature. Slides again were washed in PBS twice,for 2 minutes per wash. After dehydration for 1 minute in each of 70%,85%, and 100% ethanol, slides were used immediately, or stored at roomtemperature in the dark.

[0040] Three multicolor probe sets: A, B, and C were used in the initialhybridizations. Probe sets A-C contained the centromeric/locus specificprobes shown in Table 1. The color of the fluorophore used to label eachprobe also is shown in Table 1. Chromosomal probes (CEP®, chromosomalenumeration probe) were obtained from Vysis, Inc. (Downers Grove, Ill.).An aqua filter was used to visualize chromosomes 17 and 18. A yellowfilter was used to visualize the 9p21 locus specific probe, and a dualred/green filter or individual red or green filters were used tovisualize chromosomes 3, 7, 8, 9, 11, and Y. TABLE 1 FISH Probe SetsSpectrum Spectrum Spectrum Spectrum Probe Set Aqua Green Red Gold A 17 7 9 9p21 B 18 8 11 C 3 Y

[0041] Hybridization was performed by a HYBrite method or a conventionalmethod. In the HYBrite method, a HYBrite™ system from Vysis, DownersGrove, Ill., was used. Slides were placed on the HYBrite, and about 10μl of the probe set were added, covered, and sealed. The HYBrite wasprogrammed as follows: 73° C. for 5 min, then 37° C. for 16 hours.Slides then were washed in 0.4×SSC (0.06 M sodium chloride/0.006 Msodium citrate)/0.3% NP-40 for 2 minutes at 73° C., rinsed in 2×SSC/0.1% NP40 at room temperature, and air dried. Slides were counterstainedwith approximately 10 μl of DAPI II (125 ng/ml of4,6-diamidino-2-phenylindole dihydrochloride).

[0042] In the conventional method (i.e., Coplin jar method), a mastermix containing chromosome probes was prepared in hybridization buffercontaining 50% formamide, 2×SSC, 0.5 μg/ml Cot1 DNA, and 2 μg/ml HP DNA.The probe mix was denatured at 73° C. for 5 minutes, and slides weredenatured in denaturation buffer (70% formamide, 2×SSC) in a Coplin jarat 73° C. for 5 min (6-8 slides/jar). Slides were rinsed in each of 70%,85%, and 100% ethanol for 1 minute. Approximately 10 μl of hybridizationmix were applied to each slide, covered with a coverslip, and sealedwith rubber cement. Hybridization was performed in a humidified chamberovernight at 37° C. Slides were washed in 0.4×SSC/0.3% NP-40 at 73° C.for 2 minutes, then rinsed in 2×SSC/0.1% NP-40 briefly at roomtemperature. After air drying, slides were counterstained with DAPI II.Samples were enumerated by recording the number of FISH signals in 100consecutive cells.

[0043] In summary, this Example provides methods for isolating cellsfrom a biological sample, and hybridizing a set of chromosomal probes tothe cells.

Example 2 Detection of Bladder Cancer

[0044] Table 2 provides a summary of samples analyzed via cytology.Overall, urine cytology was positive in 13 out of 21 cancer cases,equaling a sensitivity of 62%. This value is consistent with publishedliterature. Cytology detected tumor cells in 10 out of 11 bladder cancerpatients with advanced stages of the disease (first 11 entries in Table2). Among 10 patients with superficial bladder cancer (last 10 entriesin Table 2), cytology was positive for 3 patients, equivocal for 3patients, and negative for 4 patients. ND refers to non-determined.Equivocal (E) refers to samples that were suspicious, but not diagnosticof transitional cell carcinoma (TCC). pTis refers to carcinoma in situ,and pTa refers to non-invasive papillary carcinoma. pT1 refers toinvasion of subepithelial connective tissue by the tumor; pT2 refers toinvasion of muscle by the tumor; pT3 refers to invasion of perivesicaltissue by tumor; and pT4 refers to invasion of the prostate, uterus,vagina, pelvic wall, or abdominal wall.

[0045] The percent of aneusomic cells with four or more chromosomesignals permitted the greatest discrimination between the cancer groupand normal cases. Single copy gains or losses among the control groupshowed much higher frequencies and did not result in comparablesensitivities and specificities. The percentage of aneusomic cells,using this definition, was determined for each group of samples (cancerand normal) and individually in each group. Using this definition,samples were analyzed in the following manner.

[0046] Discriminate analysis was performed to determine how well eachprobe differentiated cancer cases from normal control, using the formula${{Discrimination}\quad {Value}} = \frac{\left( {{M1} - {M2}} \right)^{2}}{{SD1}^{2} + {SD2}^{2}}$

[0047] In this formula, M1 and M2 are the mean percent aneusomic cellswith four or more signals for cancer (n=21) and normal (n=9) groups,respectively, and SD1 and SD2 are standard deviations for each group ofsamples. Table 3 provides a summary of the results for the chromosomeenumeration probes (CEP) using this analysis. TABLE 2 Detection ofBladder Cancer Via Cytology Patient Status Stage Grade Cytology 215Cancer ND ND positive 219 Cancer pT3 3 positive 224 Cancer pTis 3positive 228 Cancer ND ND ND 236 Cancer pTis 3 positive  66 Cancer pT3 3positive D Cancer ND ND positive 229 Cancer pT3 3 positive 171 CancerpT4 3 positive 227 Cancer pT4 3 positive 240 Cancer pT3 3 negative 191Cancer pTa 2 positive 225 Cancer pTa 2 positive 230 Cancer pTa 3positive 223 Cancer pTa 2 E 234 Cancer pTa 2 E 235 Cancer pTa 2 E 239Cancer pTa 3 negative  69 Cancer pTa 2 negative 110 Cancer pT1 3negative  95 Cancer pT1 3 negative

[0048] TABLE 3 Probe Discrimination Values CANCER NORMAL Mean CEP 1724.77 0.89 SD (CEP 17) 25.44 1.76 Discrimination 0.88 Mean CEP 9 15.551.07 SD (CEP 9) 19.33 2.01 Discrimination 0.55 Mean CEP 7 26.58 1.07 SD(CEP 7) 24.12 1.82 Discrimination 1.11 Mean CEP 18 13.09 1.63 SD (CEP18) 14.35 1.80 Discrimination 0.63 Mean CEP 11 16.32 1.52 SD (CEP 11)19.44 1.87 Discrimination 0.57 Mean CEP 8 15.58 1.52 SD (CEP 8) 15.471.87 Discrimination 0.81 Mean CEP 3 27.91 0.49 SD (CEP 3) 26.33 0.78Discrimination 1.08

[0049] A discrimination value of >1.0, the 95% confidence intervalaround which two populations are separated, was considered good(assuming normal distribution and roughly equivalent standard deviationvalues). By this criteria, the best probes were 7, 3, and 17. Thisanalysis provides information regarding the sensitivity and specificityof individual probes but does not reveal the sensitivity/specificity ofdifferent probe combinations.

[0050] A cutoff of two standard deviations above the mean % of aneusomiccells in normal cells was used as the criterion of FISH positivity forchromosomal gains. On the other hand, 9p21 changes in bladder cancer aremanifested as a loss and thus were evaluated as nullisomy. The cutoffvalue used for nullisomy was 3 standard deviations greater than the meanof normal samples. The percent aneusomic cells for each case by probeare shown in Table 4A for normal individuals and in Table 4B for cancerpatients. In Table 4B, samples below the double line are cytology falsenegative cases, and shaded text indicates false negative FISH resultsusing the cutoff values from Table 3. As shown in Table 4A, the percentof aneusomic cells (as defined by chromosomal gains) in normal samplesis low, ranging from about 0.5% to about 4.5%. TABLE 4A Percent ofaneusomic cells: normal cases CEP CEP CEP CEP CEP CEP CEP Patient 17 9 718 11 8 3 9p21 2F 0 0 0 0 0 0 0 0 3M 0 0 0 0 0 0 0 1.00 8M 0 0 0 0 0 0 00 9M 0 0 0 5 4 5 1 3.00 F5 4 2 2 2 1 1 0 ND F6 0 6 0 0 0 0 1.43 0 M12 41.67 6 3 5 4 2.00 14.00 M28 0 0 1.67 1.67 1.67 1.67 0 3.33 M23 0 1.07 03 2.00 2.00 0 15.00 MEAN 0.88 1.81 1.07 1.63 1.51 1.51 0.49 4.54 SD 1.762.01 1.81 1.79 1.87 1.87 0.77 6.29 Mean + 4.41 5.09 4.7 5.2 5.26 5.262.05 17.12 2D

[0051] TABLE 4B Percent of aneusomic cells: cancer cases CEP CEP CEP CEPCEP CEP CEP Null Patient 17 9 7 18 11 8 3 9p21 171 21 0 24 2 42 44 1 9191 3.45 10.34 17.24 44.9 26.53 38.78 50 4.76 215 38 21 24 5.81 4.652.33 60.32 13 219 39 44 41 14 11 24 53 10 224 73.68 70.3 81.05 ND ND NDND 69.47 225 5 4 9 5 12 6 20 16 227 52 33 69 37 14 34 44 1 228 34 13 2932 2 34 53 3 229 3 20 35 5 47 8 43 4 230 38 39 32 13 8 9 44 8 236 50 1749 21 34 35 54 62 66 84 5 66 36 70 7 78.89 27 D 41.94 38.71 38.71 ND NDND ND 12.9 239 0 0 0 3 4 4 0 20 240 5 0 13 18 23 32 0 1 69 2 1 2 0 0 0 52 95 0 0 0 2 2 2 0 2 110 10.1 1 18.18 3 2 6 11 20.2 223 0 0 0 3 5 7 0 43234 11 9 9 3 2 2 3 3 235 9 0 1 1 1 1 10 1

[0052] TABLE 5 Results of FISH and Cytology for Normal and BladderCancer Patients Bladder Cancer (n = 21) Non Cancer (n = 3) Cytology+Cytology− Cytology− FISH + 13 7 0 FISH −  0 1 3

[0053] Overall, as shown in Table 4B, chromosome 7 demonstrated thehighest sensitivity of any individual probe (76%, 16/21). Probes tochromosome 3 and 9p21 complemented chromosome 7 in that they werepositive for some of the cases with normal chromosome 7 results. Incombination, the three probes to chromosomes 3, 7 and 9p21 detected20/21 bladder cancer cases and more importantly 7/8 cytology falsenegative cases (Table 4B). In this data set, this equates to an overallsensitivity of 95%.

[0054] The chromosome 17 probe (along with the chromosome 3 probe)detected the highest number of cytology false-negative samples (4/8).

Example 3 Comparative Analysis with Standard Screening Methods

[0055] 190 patients from the Mayo clinic were prospectively enrolled inthis study. A majority of the patients either had a previous diagnosisof bladder cancer or were being evaluated for a possible initialdiagnosis of bladder cancer (e.g. for microhematuria). A smallproportion of the patients were being evaluated for genitourinarydisorders other than bladder cancer. Test methodologies comparedincluded urine cytology, cystoscopy, BTA STAT (C.R Bard, Inc., MurrayHill, N.J.), FISH, and hemoglobin dipstick (Bayer Corporation,Diagnostic Division, Elkhart, Ind.).

[0056] FISH generally was performed as described in Example 1.Approximately 25 to 200 ml of urine were collected per patient. Urinesamples were stored at 4° C. for up to 48 hours, and processed bycentrifugation at 600 g for 10 minutes. The supernatant was discarded,and the pellet was resuspended in 25-50 ml of 1×phosphate-bufferedsaline (PBS). After centrifugation for 5 minutes at 600 g, thesupernatant again was discarded. Pellets were resuspended slowly in1.5-5 ml of fixative (methanol:glacial acetic acid, 3:1), andcentrifuged for 5 minutes at 600 g. The fixative was carefully removed,and this step was repeated two more times.

[0057] After the final centrifugation, the fixative was removed, leavingan appropriate amount of the solution for dropping onto slides such asthe 12-Circle 6 mm Shandon Lipshaw slides. If the cell pellet was verysmall, and hardly visible to the eye, approximately 100 μl were leftabove the pellet. If the cell pellet was easily visible by eye, as muchCarnoy's fixative as possible was removed, and 0.5 ml of fresh Carnoy'sfixative was added. If no pellet was visible, the entire sample oftenwas dropped on one slide. Approximately 3, 10, and 30 μl of the cellsuspension were then dropped into three separate 0.6 mm wells of aShandon 12 well slide. The cellularity (i.e. density of cells) in thesewells was assessed with a phase contrast microscope. If the wellcontaining the smallest volume of cell suspension is too dense, the cellsuspension is further diluted and a portion of this dilution is put in afourth well. If the well containing the greatest volume of cellsuspension does not have enough cells, the cell suspension isconcentrated and placed in a fourth well.

[0058] Slides containing the samples were incubated in 2×SSC at 37° C.for 10-30 minutes, then incubated in 0.2 mg/ml pepsin for 10 minutes at37° C. Slides then were washed in PBS twice, for 2 minutes per wash, atroom temperature. Cells were fixed in 2.5% Neutral Buffered Formalin for5 minutes at room temperature. Slides again were washed in PBS for 5minutes. After dehydration for 1 minute in each of 70%, 85%, and 100%ethanol, slides were dried on a slide warmer at 45-50° C. for 2 minutes.Slides were either left in 100% ethanol at 4° C. until further use, orwere used for FISH.

[0059] Hybridization was performed by a HYBrite method or a conventionalmethod. In the HYBrite method, a HYBrite™ system from Vysis, DownersGrove, Ill., was used. Slides were placed on the HYBrite, and about 3 μlof probe were added per target, then the slides were covered and sealed.The HYBrite was programmed as follows: 73° C. for 5 min, then 37° C. for16 hours. Slides then were washed in 0.4 SSC (0.06 M sodiumchloride/0.006 M sodium citrate)/0.3% NP-40 for 2 minutes at 73° C.,rinsed in 2×SSC/0.1% NP40 at room temperature, and air dried. Slideswere counterstained with approximately 3 μl of DAPI II (125 ng/ml of4,6-diamidino-2-phenylindole dihydrochloride).

[0060] In the conventional method, a master mix containing chromosomeprobes was prepared in hybridization buffer containing 50% formamide,2×SSC, 0.5)g/ml Cot1 DNA, and 2 μg/ml HP DNA. The probe mix wasdenatured at 73° C. for 5 minutes, and slides were denatured indenaturation buffer (70% formamide, 2×SSC) in a Coplin jar at 73° C. for5 min (6-8 slides/jar). Slides were rinsed in each of 70%, 85%, and 100%ethanol for 1 minute. Samples were dried on a slide warmer for 2 minutesor less. Approximately 3 μl of hybridization mix were applied per targeton each slide, and the slides were covered immediately with a coverslip,which then was sealed with rubber cement. Hybridization was performed ina humidified chamber overnight at 37° C. Slides were washed in0.4×SSC/0.3% NP-40 at 73° C. for 2 minutes, then rinsed in 2×SSC/0.1%NP-40 briefly at room temperature. After air drying, slides werecounterstained with DAPI II. Twenty cytologically abnormal cells wereselected in each sample, and analyzed for their FISH pattern.

[0061] For 53 cancer cases, as diagnosed by biopsy (Stage/Grade) (n=49)and cytology positive TCC (n=34), cytology had a sensitivity of 57%(equivocal cytology diagnoses counted as positive), cystoscopy had asensitivity of 88% (equivocal cystoscopy results counted as positive),BTA STAT had a sensitivity of 71%, and FISH had a sensitivity of 86%. In63 cancer negative cases, as indicated by a negative cystoscopy with orwithout negative cytology and no history of bladder cancer, BTA STAT hada specificity of 73%, and FISH had a specificity of 90% (specificity ofcystoscopy and cytology cannot be determined since they were used todefine which patients did not have bladder cancer.

[0062] Two of the three patients with “false positive” FISH results alsohad positive telomerase, hemoglobin dipstick and BTA-STAT or BTA-TRAKresults. Should these two patients prove to have bladder cancer, thespecificity of FISH will approach 100%. This has implications for thepositive predictive value of the test as discussed below. Table 6 showsthe sensitivity of the various tests by stage and grade. In Table 6, “E”refers to equivocal, and “*” indicates 3 FISH⁺ samples were cancer bycytology only and were not included.

[0063] The positive and negative predictive values (PV), based on adisease prevalence of 28% (53/190), specificity of 93% and sensitivityof 77%, are indicated in Table 7. TABLE 6 Grade 1 Grade 2 Grade 3Overall Cytology pTa 1+[1E]/8 5/13 2/2 39% (9/23) pT1-pT4 3+[3E]/9 66%(6/9) pTis 5+[5E]/12 83% (10/12) Overall 25% (2/8) 38% (5/13) 78%(18/23) 57% (25/44) Cystoscopy pTa 5+[1E]/7 13/13 2/2 95% (21/22)pT1-pT4 4+[3E]/9 78% (7/9) pTis 5+[5E]/12 83% (10/12) Overall 86% (6/7)100% (13/13) 83% (19/23) 88% (38/43) FISH pTa 4/7 9/12 2/2 71% (15/21)pT1-pT4 12/12 100% (12/12) pTis 10/10 100% (10/10) Overall 57% (4/7) 75%(9/12) 100% (24/24) 86% (37/43) BTA STAT pTa 4/8 6/14 2/2 50% (12/24)pT1-pT4 9/10 90% (9/10) pTis 12/12 100% (12/12) Overall 50% (4/8) 43%(6/14) 96% (24/25) 71% (33/46)

[0064] TABLE 7 Predictive Value Method PV+ PV− FISH 77% 94% BTA STAT 51%87%

[0065] Fifty-one out of the 53 cancer cases had a dipstick result. Thesensitivity of the hemoglobin dipstick test was 91% for stages pT1-pT4and 100% for carcinoma in situ (Tis), making it an ideal screening test(Table 8). However, the specificity (52%) of the hemoglobin dipsticktest was poor. While the high sensitivity and low cost of the hemoglobindipstick make it a useful screening test, it is clear that positiveresults must be confirmed by a more specific test. Cytology cannot bethe test of choice because it detected as positive only 33% of pT2-pT4cases and missed 2 of 12 carcinoma in situ (see Table 6). TABLE 8 Hbdipstick Trace to 3+ Negative Sensitivity pTa 13 11  54% pT1-pT4 10 191% pTis 12 0 100% 

[0066] The clinical utility of FISH was compared to the standardcytology/cystoscopy testing regimen. Of the 53 cancer cases, there were22 cases of advanced stage disease (pT2-pT4 or pTIS). Failure to detectthese cases represents the most serious false negative situation. Whilecytology/cystoscopy was positive in 13/22 cases for a sensitivity of59%, FISH had 100% sensitivity, i.e., was able to detect 22/22 cases(see Table 9 for a representation of 5 of these cases). This indicatesthat FISH is an improved cytology test which has high sensitivity fordetecting advance stages of disease. TABLE 9 Patient Stage GradeCystoscopy Cytology FISH 79 pT2 Grade 3 E E Pos 132 pT2 Grade 3 Neg NegPos 1 pT3 Grade 3 Neg Neg Pos 115 pT4 Grade 3 E E Pos 180 pTIS Grade 3 ENeg Pos

Other Embodiments

[0067] It is understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of screening for cancer in a subjectcomprising: (a) hybridizing a set of chromosomal probes to a biologicalsample from said subject; (b) selecting cells from said biologicalsample; (c) determining the presence or absence of aneusomic cells insaid selected cells; (d) correlating presence of aneusomic cells in theselected cells with cancer in said subject.
 2. The method of claim 1,wherein said biological sample is selected from the group consisting ofurine, blood, cerebrospinal fluid, pleural fluid, sputum, peritonealfluid, bladder washings, oral washings, tissue samples, touch preps, andfine-needle aspirates.
 3. The method of claim 1, wherein said biologicalsample is concentrated.
 4. The method of claim 1, wherein saidbiological sample is urine.
 5. The method of claim 1, wherein saidcancer is selected from the group consisting of bladder cancer, lungcancer, breast cancer, ovarian cancer, prostate cancer, colorectalcancer, renal cancer, and leukemia.
 6. The method of claim 1, whereinsaid cancer is bladder cancer.
 7. The method of claim 1, wherein saidchromosomal probes are fluorescently labeled.
 8. The method of claim 1,wherein said set comprises at least three chromosomal probes.
 9. Themethod of claim 1, wherein said set comprises at least one centromericprobe.
 10. The method of claim 9, wherein said set further comprises atleast one locus specific probe.
 11. The method of claim 9, wherein saidcentromeric chromosomal probes are selected from the group consisting ofchromosomal probes 3, 7, 8, 11, 15, 17, 18, and Y.
 12. The method ofclaim 10, wherein said locus specific probe is 9p21.
 13. The method ofclaim 8, wherein said set comprises chromosomal probes 3, 7, and
 17. 14.The method of claim 13, wherein said set further comprises locusspecific probe 9p21.
 15. The method of claim 1, wherein cells areselected by nuclear morphology.
 16. The method of claim 1, wherein cellsare selected by nuclear size.
 17. The method of claim 1, wherein cellsare selected by shape of nucleus.
 18. The method of claim 15, whereinnuclear morphology is assessed by DAPI staining.
 19. A set ofchromosomal probes, said set comprising centromeric probes tochromosomes 3, 7, and
 17. 20. The set of chromosomal probes of claim 19,said set further comprising a locus-specific probe.
 21. The set ofchromosomal probes of claim 20, wherein said locus-specific probe is9p21.
 22. A kit for detecting cancer, said kit comprising a set ofchromosomal probes, wherein said set comprises centromeric probes tochromosomes 3, 7, and
 17. 23. The kit of claim 22, said kit furthercomprising a locus-specific probe.
 24. The kit of claim 23, wherein saidlocus-specific probe is 9p21.
 25. The kit of claim 22, wherein saidchromosomal probes are fluorescently labeled.